RESUMEN

Lens fiber cell gap junctions contain alpha(3) (Cx46) and alpha(8) (Cx50) connexins. To examine the roles of the two different connexins in lens physiology, we have genetically engineered mice lacking either alpha(3) or alpha(8) connexin. Intracellular impedance studies of these lenses were used to measure junctional conductance and its sensitivity to intracellular pH. In Gong et al. 1998, we described results from alpha(3) connexin knockout lenses. Here, we present original data from alpha(8) connexin knockout lenses and a comparison with the previous results. The lens has two functionally distinct domains of fiber cell coupling. In wild-type mouse lenses, the outer shell of differentiating fibers (see 1, DF) has an average coupling conductance per area of cell-cell contact of approximately 1 S/cm(2), which falls to near zero when the cytoplasm is acidified. In the inner core of mature fibers (see 1, MF), the average coupling conductance is approximately 0.4 S/cm(2), and is insensitive to acidification of the cytoplasm. Both connexin isoforms appear to contribute about equally in the DF since the coupling conductance for either heterozygous knockout (+/-) was approximately 70% of normal and 30-40% of the normal for both -/- lenses. However, their contribution to the MF was different. About 50% of the normal coupling conductance was found in the MF of alpha(3) +/- lenses. In contrast, the coupling of MF in the alpha(8) +/- lenses was the same as normal. Moreover, no coupling was detected in the MF of alpha(3) -/- lenses. Together, these results suggest that alpha(3) connexin alone is responsible for coupling MF. The pH- sensitive gating of DF junctions was about the same in wild-type and alpha(3) connexin -/- lenses. However, in alpha(8) -/- lenses, the pure alpha(3) connexin junctions did not gate closed in the response to acidification. Since alpha(3) connexin contributes about half the coupling conductance in DF of wild-type lenses, and that conductance goes to zero when the cytoplasmic pH drops, it appears alpha(8) connexin regulates the gating of alpha(3) connexin. Both connexins are clearly important to lens physiology as lenses null for either connexin lose transparency. Gap junctions in the MF survive for the lifetime of the organism without protein turnover. It appears that alpha(3) connexin provides the long-term communication in MF. Gap junctions in DF may be physiologically regulated since they are capable of gating when the cytoplasm is acidified. It appears alpha(8) connexin is required for gating in DF.

Asunto(s)

Comunicación Celular , Conexinas/fisiología , Uniones Comunicantes/fisiología , Cristalino/química , Animales , Concentración de Iones de Hidrógeno , Activación del Canal Iónico , Ratones , Ratones Noqueados

RESUMEN

The antibody 38C2 efficiently catalyzed a retro-Michael reaction to convert a novel, cell-permeable fluorogenic substrate into fluorescein within living cells. In vitro, the antibody converted the substrate to fluorescein with a k(cat) of 1.7 x 10(-5) s(-1) and a catalytic proficiency (k(cat)/k(uncat)K(m)) of 1.4 x 10(10) m(-1) (K(m) = 7 microm). For hybridoma cells expressing antibody or Chinese Hamster Ovarian (CHO) cells injected with antibody, incubation of the substrate in the extracellular medium resulted in bright intracellular fluorescence distinguishable from autofluorescence or noncatalyzed conversion of substrate. CHO cells loaded with antibody were 12 times brighter than control cells, and more than 85% of injected cells became fluorescent. The fluorescein produced by the antibody traveled into neighboring cells through gap junctions, as demonstrated by blocking dye transfer using the gap junction inhibitor oleamide. The presence of functional gap junctions in CHO cells was confirmed through oleamide inhibition of lucifer yellow transfer. These studies demonstrate the utility of the intracellular antibody reaction, which could generate tracer dyes in specific cells within complex multicellular environments simply by bathing the system in substrate.

Asunto(s)

Anticuerpos Catalíticos/metabolismo , Comunicación Celular/fisiología , Fluoresceína/metabolismo , Fluoresceínas/metabolismo , Colorantes Fluorescentes/metabolismo , Fructosa-Bifosfato Aldolasa/metabolismo , Uniones Comunicantes/metabolismo , Fragmentos Fab de Inmunoglobulinas/metabolismo , Animales , Anticuerpos Catalíticos/química , Células CHO , Cricetinae , Fluoresceína/química , Fluoresceínas/química , Colorantes Fluorescentes/química , Microinyecciones , Estructura Molecular

RESUMEN

Disruption of the connexin alpha 3 (Cx46) gene (alpha 3 (-/-)) in mice results in severe cataracts within the nuclear portion of the lens. These cataracts are associated with proteolytic processing of the abundant lens protein gamma-crystallin, leading to its aggregation and subsequent opacification of the lens. The general cysteine protease inhibitor, E-64, blocked cataract formation and gamma-crystallin cleavage in alpha 3 (-/-) lenses. Using a new class of activity-based cysteine protease affinity probes, we identified the calcium-dependent proteases, m-calpain and Lp82, as the primary targets of E-64 in the lens. Profiling changes in protease activities throughout cataractogenesis indicated that Lp82 activity was dramatically increased in alpha 3 (-/-) lenses and correlated both spatially and temporally with cataract formation. Increased Lp82 activity was due to calcium accumulation as a result of increased influx and decreased outflux of calcium ions in alpha 3 (-/-) lenses. These data establish a role for alpha 3 gap junctions in maintaining calcium homeostasis that in turn is required to control activity of the calcium-dependent cysteine protease Lp82, shown here to be a key initiator of the process of cataractogenesis.

Asunto(s)

Calpaína/metabolismo , Catarata/fisiopatología , Comunicación Celular/fisiología , Conexinas/fisiología , Cisteína Endopeptidasas/metabolismo , Uniones Comunicantes/fisiología , Cristalino/fisiología , Leucina/análogos & derivados , Leucina/farmacología , Animales , Calcio/metabolismo , Catarata/genética , Catarata/patología , Catarata/prevención & control , Conexinas/deficiencia , Conexinas/genética , Inhibidores de Cisteína Proteinasa/farmacología , Cristalino/efectos de los fármacos , Cristalino/patología , Ratones , Ratones Endogámicos , Ratones Noqueados , Técnicas de Cultivo de Órganos

RESUMEN

Because in mammals the anterior pituitary lacks innervation, we investigated whether gap junctions established between selected cells within the gland are part of an intrapituitary mechanism to ensure physiological synchronization of cells involved in the control of hormone secretion. We report here the dynamics of anterior pituitary connexin 43 (Cx43)-gap junctions throughout the mink (Mustela vison) annual reproductive cycle and its relationship with the anterior pituitary prolactin (PRL) content that parallels variations in serum PRL levels documented in the literature. We found that PRL anterior pituitary levels were maximal in spring and during lactation and that they were minimal in autumn and winter. Anterior pituitary Cx43 levels were maximal during periods of high PRL secretion. During these periods, Cx43-positive gap junctions localized to stellate-shaped cells occupying the center of anterior pituitary follicles and to the rounded cells occupying the remaining follicles. Connexin 43-positive gap junctions were also observed between adjacent follicles. During periods of low PRL pituitary content, Cx43-positive gap junctions localized to the stellate cells but not to the cells of the remaining follicles. Moreover, Cx43 labeling was undetected between adjacent follicles. To assess between which cells within the mink anterior pituitary the Cx43 gap junctions were established, the different anterior pituitary cell populations were separated by a discontinuous Percoll gradient, and Western blot analyses of each cell population using Cx43 antibodies were performed. The immunoblots showed a Cx43 immunoreactive band associated with the cell layer enriched in S-100-positive, stellate-shaped cells. The result was confirmed by fluorescence microscopy studies that showed that Cx43-mediated gap junctions were established preferentially between the cultured S-100-positive, elongated cells. The results show that in mink stellate cells, the junctional machinery associated with the Cx43 protein varies in synchrony with the anterior pituitary PRL content throughout the mink annual reproductive cycle. It is suggested that the Cx43 gap junctions on the stellate cells play an important role in the synchronization of cellular activity within selected follicles of the anterior pituitary, thus contributing to the control of PRL secretion during the annual reproductive cycle.

Asunto(s)

Conexina 43/metabolismo , Visón/metabolismo , Adenohipófisis/metabolismo , Prolactina/metabolismo , Estaciones del Año , Animales , Células Cultivadas , Electroforesis , Femenino , Hormona Folículo Estimulante/metabolismo , Hormona del Crecimiento/metabolismo , Immunoblotting , Técnicas para Inmunoenzimas , Inmunohistoquímica , Masculino , Microscopía Fluorescente , Adenohipófisis/citología , Proteínas S100/metabolismo

RESUMEN

We used electron cryo-microscopy and image analysis to examine frozen-hydrated, two-dimensional (2D) crystals of a recombinant, 30-kDa C-terminal truncation mutant of the cardiac gap junction channel formed by 43-kDa alpha(1) connexin. To our knowledge this is the first example of a structural analysis of a membrane protein that has been accomplished using microgram amounts of starting material. The recombinant alpha(1) connexin was expressed in a stably transfected line of baby hamster kidney cells and spontaneously assembled gap junction plaques. Detergent treatment with Tween 20 and 1,2-diheptanoyl-sn-phosphocholine resulted in well-ordered 2D crystals. A three-dimensional density (3D) map with an in-plane resolution of approximately 7.5 A revealed that each hexameric connexon was formed by 24 closely packed rods of density, consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. In the extracellular gap the aqueous channel was bounded by a continuous wall of protein that formed a tight electrical and chemical seal to exclude exchange of substances with the extracellular milieu.

Asunto(s)

Conexina 43/ultraestructura , Uniones Comunicantes/ultraestructura , Canales Iónicos/ultraestructura , Animales , Células Cultivadas , Conexina 43/genética , Cricetinae , Microscopía por Crioelectrón , Cristalización , Técnica del Anticuerpo Fluorescente , Procesamiento de Imagen Asistido por Computador , Modelos Moleculares , Mutación , Estructura Secundaria de Proteína , Ratas , Proteínas Recombinantes/ultraestructura , Transfección

RESUMEN

A series of arachidonic acid amides including anandamide and arachidonamide that act as potent inhibitors of the rat glial cell gap junction is described.

Asunto(s)

Ácido Araquidónico/síntesis química , Comunicación Celular/fisiología , Uniones Comunicantes/fisiología , Animales , Modelos Químicos , Neuroglía/efectos de los fármacos , Ratas

RESUMEN

Gap junctions in the heart play an important functional role by electrically coupling cells, thereby organizing the pattern of current flow to allow co-ordinated muscle contraction. Cardiac gap junctions are therefore intimately involved in normal conduction as well as the genesis of potentially lethal arrhythmias. We recently utilized electron cryo-microscopy and image analysis to examine frozen-hydrated 2D crystals of a recombinant, C-terminal truncated form of connexin 43 (Cx43; alpha 1), the principal cardiac gap junction protein. The projection map at 7 A resolution revealed that each 30 kDa connexin subunit has a transmembrane alpha-helix that lines the aqueous pore and a second alpha-helix in close contact with the membrane lipids. The distribution of densities allowed us to propose a model in which the two apposing connexons that form the channel are staggered by approximately 30 degrees. We are now recording images of tilted, frozen-hydrated 2D crystals, and a preliminary 3D map has been computed at an in-plane resolution of approximately 7.5 A and a vertical resolution of approximately 25 A. As predicted by our model, the two apposing connexons that form the channel are staggered with respect to each other for certain connexin molecular boundaries within the hexamer. Within the membrane interior each connexin subunit displays four rods of density, which are consistent with an alpha-helical conformation for the four transmembrane domains. Preliminary studies of BHK hamster cells that express the truncated Cx43 designated alpha 1 Cx263T demonstrate that oleamide, a sleep inducing lipid, blocks in vivo dye transfer, suggesting that oleamide causes closure of alpha 1 Cx263T channels. The comparison of the 3D structures in the presence and absence of oleamide may provide an opportunity to explore the conformational changes that are associated with oleamide-induced blockage of dye transfer. The structural details revealed by our analysis will be essential for delineating the molecular basis for intercellular current flow in the heart, as well as the general molecular design and functional properties of this important class of channel proteins.

Asunto(s)

Conexina 43/ultraestructura , Microscopía por Crioelectrón/métodos , Secuencia de Aminoácidos , Animales , Conexina 43/metabolismo , Cricetinae , Humanos , Datos de Secuencia Molecular , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura

RESUMEN

Connexin alpha 3 (Cx46 or Gja3) gene targeted null mice developed lens nuclear cataracts shortly after birth. A large variance in the cataracts was observed in alpha 3 null sibs on a mixed 129SvJae X C57BL/6J F3 background. This suggested that the genetic background might influence the cataract phenotype. Therefore, we placed the alpha 3 null mutation into a 129SvJae background, and also backcrossed the mutation for six generations into 129SvJ and C57BL/6J backgrounds. While alpha 3 nulls on the two 129 backgrounds contained severe cataracts associated with gamma crystallin cleavage, alpha 3 nulls on the C57B16 background had far milder cataracts with no detectable gamma crystallin cleavage. These findings suggest that a genetic modifier exists that influences gamma crystallin stability, and that gamma crystallin breakdown is associated with severe nuclear cataracts.

Asunto(s)

Catarata/genética , Conexinas/genética , Cristalinas/metabolismo , Cristalino/química , Animales , Western Blotting , Catarata/metabolismo , Conexinas/fisiología , Cruzamientos Genéticos , Cristalinas/análisis , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Fenotipo

RESUMEN

Gap junction membrane channels mediate electrical and metabolic coupling between adjacent cells. The structure of a recombinant cardiac gap junction channel was determined by electron crystallography at resolutions of 7.5 angstroms in the membrane plane and 21 angstroms in the vertical direction. The dodecameric channel was formed by the end-to-end docking of two hexamers, each of which displayed 24 rods of density in the membrane interior, which is consistent with an alpha-helical conformation for the four transmembrane domains of each connexin subunit. The transmembrane alpha-helical rods contrasted with the double-layered appearance of the extracellular domains. Although not indicative for a particular type of secondary structure, the protein density that formed the extracellular vestibule provided a tight seal to exclude the exchange of substances with the extracellular milieu.

Asunto(s)

Conexina 43/química , Uniones Comunicantes/química , Miocardio/química , Estructura Secundaria de Proteína , Animales , Línea Celular , Cricetinae , Cristalografía , Uniones Comunicantes/ultraestructura , Membrana Dobles de Lípidos/química , Modelos Moleculares , Mutación , Miocardio/ultraestructura , Conformación Proteica , Proteínas Recombinantes/química

RESUMEN

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in alpha1 connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for alpha1 connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing alpha1 connexins, and from alpha1 connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative alpha1 connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and alpha1 connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous alpha1 connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and alpha1 connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the alpha1 connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium- an elevation in the CMV43 mice vs. a reduction in the alpha1 connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non-neural crest- derived tissues. Overall, these findings suggest that gap junction communication mediated by alpha1 connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of alpha1 connexin function.

Asunto(s)

Comunicación Celular/fisiología , Conexina 43/fisiología , Uniones Comunicantes/fisiología , Cresta Neural/fisiología , Neuronas/fisiología , Animales , División Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Conexina 43/deficiencia , Conexina 43/genética , Femenino , Corazón Fetal/inervación , Uniones Comunicantes/ultraestructura , Dosificación de Gen , Genotipo , Heterocigoto , Homocigoto , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Cresta Neural/citología , Neuronas/citología , Neuronas/efectos de los fármacos , Ácidos Oléicos/farmacología , Técnicas de Cultivo de Órganos , Proteínas Recombinantes de Fusión/biosíntesis

RESUMEN

Fiber cells of the lens are interconnected by an extensive network of gap junctions containing alpha3 (Cx46) and alpha8 (Cx50) connexins. A specific role for these connexins in lens homeostasis is not known. To determine the contribution of these connexins to lens function, we used impedance techniques to study cell-to-cell coupling in lenses from homozygous alpha3 knockout (-/-), heterozygous (+/-), and wild-type (+/+) mice. Western blots and immunofluorescence data indicated that alpha8 remained at similar levels in the three classes of lenses, whereas alpha3 was approximately 50% of the normal level in the +/- lenses, and it was absent from the -/- lenses. Moreover, the data from +/+ lenses suggest that a cleavage of connexins occurs abruptly between the peripheral shell of differentiating fibers (DF) and the inner core of mature fibers (MF). The appearance of the cleaved connexins was correlated to a change in the coupling conductance. In -/- lenses the coupling conductance of MF was zero, and these fibers were depolarized by about 30 mV from normal (approximately -65 mV). The DF remained coupled, but the conductance was reduced to 30-35% of normal. However, the gap junctions in the DF of alpha3 -/- lenses remained sensitive to pH. We conclude that alpha3 connexin is necessary for the coupling of central fibers to peripheral cells, and that this coupling is essential for fiber cell homeostasis because uncoupled MF depolarize and subsequently become opaque.

Asunto(s)

Conexinas/fisiología , Uniones Comunicantes/fisiología , Cristalino/fisiología , Animales , Conexinas/química , Conexinas/deficiencia , Electrofisiología , Heterocigoto , Concentración de Iones de Hidrógeno , Inmunohistoquímica , Cinética , Cristalino/citología , Ratones , Ratones Noqueados

RESUMEN

Oleamide is an endogenous fatty acid primary amide that possesses sleep-inducing properties in animals and has been shown to effect serotonergic systems and block gap junction communication in a structurally specific manner. Herein, the structural features of oleamide required for inhibition of the gap junction-mediated chemical and electrical transmission in rat glial cells are defined. The effective inhibitors fall into two classes of fatty acid primary amides of which oleamide and arachidonamide are the prototypical members. Of these two, oleamide constitutes the most effective, and its structural requirements for inhibition of the gap junction are well defined. It requires a chain length of 16-24 carbons of which 16-18 carbons appears optimal, a polarized terminal carbonyl group capable of accepting but not necessarily donating a hydrogen bond, a Delta9 cis double bond, and a hydrophobic methyl terminus. Within these constraints, a range of modifications are possible, many of which may be expected to improve in vivo properties. A select set of agents has been identified that serves both as oleamide agonists and as inhibitors of fatty acid amide hydrolase, which is responsible for the rapid inactivation of oleamide.

Asunto(s)

Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/efectos de los fármacos , Ácidos Oléicos/farmacología , Amidohidrolasas/metabolismo , Animales , Células Cultivadas , Isomerismo , Neuroglía/efectos de los fármacos , Ácidos Oléicos/química , Ácidos Oléicos/metabolismo , Ratas , Relación Estructura-Actividad

RESUMEN

We reported previously (Falk, M. M., Kumar, N. M., and Gilula, N. B. (1994) J. Cell Biol. 127, 343-355) that the membrane integration of polytopic connexin polypeptides can be accompanied by an inappropriate cleavage that generates amino-terminal truncated connexins. While this cleavage was not detected in vivo, translation in standard cell-free translation/translocation systems resulted in the complete cleavage of all newly integrated connexins. Partial cleavage occurred in heterologous expression systems that correlated with the expression level. Here we report that the transmembrane topology of connexins generated in microsomal membranes was identical to the topology of functional connexins in plasma membranes. Characterization of the cleavage site and reaction showed that the connexins were processed by signal peptidase immediately downstream of their first transmembrane domain in a reaction similar to the removal of signal peptides from pre-proteins. Increasing the length and hydrophobic character of the signal anchor sequence of connexins completely prevented the aberrant cleavage. This result indicates that their signal anchor sequence was falsely recognized and positioned as a cleavable signal peptide within the endoplasmic reticulum translocon, and that this mispositioning enabled signal peptidase to access the cleavage sites. The results provide direct evidence for the involvement of unknown cellular factors in the membrane integration process of connexins.

Asunto(s)

Membrana Celular/metabolismo , Conexinas/biosíntesis , Conexinas/química , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Animales , Conexinas/genética , ADN Complementario/genética , Expresión Génica , Proteínas de la Membrana/genética , Microsomas/metabolismo , Datos de Secuencia Molecular , Serina Endopeptidasas/química

RESUMEN

Retinoids and phorbol esters have profound effects on proliferation and differentiation of epidermal keratinocytes when applied topically on rodent skin. Since both agents also modulate gap junction (GJ)-mediated cell-cell communication, we have examined the effects of all-trans retinoic acid (RA) and 12-O-tetradecanoylphorbol-13-acetate (TPA) on the expression of alpha1 (Cx43) and beta2 (Cx26) connexins, the two major gap junction gene products in mature rat epidermis. In fully differentiated, mature epidermis, alpha1 is expressed in the lower, less differentiated portion, while beta2 is localized in upper, more differentiated layers. Dorsal skin of 21-day old rats was treated topically with a single dose of RA, TPA or vehicle alone and used for histological and molecular analyses at different time points. Keratinocytes in interfollicular epidermis were examined for proliferation and differentiation using specific antibodies for keratins (K10, K14) and proliferating cell nuclear antigen (PCNA). An increase in epidermal thickness was noticed within 4 hours after the application of RA or TPA. This increase, however, appeared to be primarily due to hypertrophy, since no substantial changes were observed in the proliferative index of epidermal keratinocytes. PCNA immunoreactivity significantly increased after 8 hours treatment of RA or TPA, suggesting a hyperproliferative growth response. Epidermal hyperplasia was confirmed by monitoring the expression patterns of K10 and K14 in RA- or TPA-treated skin. RA-induced hyperplasia lasted longer as compared to TPA induction. Changes in keratin phenotypes were paralleled by an increase in alpha1 and beta2 connexin expression as well as their colocalization in same epidermal layers. Differences in hyperplastic growth response kinetics were also confirmed at the connexin level, with beta2 antigen sustained for longer and at higher levels in suprabasal layers of RA-treated skin. Overall, this type of connexin expression resembled that observed in the non-differentiated rat epidermis during embryonic development. An increase in alpha1 and beta2 connexin abundance was also observed at the protein and RNA levels. At 96 hours after RA or TPA treatment, expression of both connexins was similar to that of the control epidermis. Taken together, these findings suggest that a higher level of GJ-mediated cell-cell communication, is required for the maintenance of homeostasis during periods of rapid epidermal growth and differentiation.

Asunto(s)

Epidermis/patología , Epidermis/fisiología , Uniones Comunicantes/fisiología , Animales , Antineoplásicos/farmacología , Carcinógenos/farmacología , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/fisiología , Conexina 26 , Conexina 43/análisis , Conexina 43/genética , Conexinas/análisis , Conexinas/genética , Epidermis/química , Uniones Comunicantes/química , Uniones Comunicantes/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Hiperplasia , Queratinocitos/química , Queratinocitos/fisiología , Queratinas/genética , ARN Mensajero/análisis , Ratas , Ratas Wistar , Acetato de Tetradecanoilforbol/farmacología , Tretinoina/farmacología

RESUMEN

Gap junction channels formed by alpha3 (Cx46) and alpha8 (Cx50) connexin provide pathways for communication between the fiber cells in the normal transparent lens. To determine the specific role of alpha3 connexin in vivo, the alpha3 connexin gene was disrupted in mice. Although the absence of alpha3 connexin had no obvious influence on the early stages of lens formation and the differentiation of lens fibers, mice homozygous for the disrupted alpha3 gene developed nuclear cataracts that were associated with the proteolysis of crystallins. This study establishes the importance of gap junctions in maintaining normal lens transparency by providing a cell-cell signaling pathway or structural component for the proper organization of lens membrane and cytoplasmic proteins.

Asunto(s)

Catarata/genética , Conexinas/deficiencia , Conexinas/genética , Cristalino/patología , Envejecimiento , Secuencia de Aminoácidos , Animales , Catarata/patología , Conexinas/química , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Cristalino/crecimiento & desarrollo , Cristalino/ultraestructura , Ratones , Ratones Noqueados , Ratones Transgénicos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Mapeo Restrictivo , beta-Galactosidasa/biosíntesis

RESUMEN

Several different gap junction channel subunit isotypes, known as connexins, were synthesized in a cell-free translation system supplemented with microsomal membranes to study the mechanisms involved in gap junction channel assembly. Previous results indicated that the connexins were synthesized as membrane proteins with their relevant transmembrane topology. An integrated biochemical and biophysical analysis indicated that the connexins assembled specifically with other connexin subunits. No interactions were detected between connexin subunits and other co-translated transmembrane proteins. The connexins that were integrated into microsomal vesicles assembled into homo- and hetero-oligomeric structures with hydrodynamic properties of a 9S particle, consistent with the properties reported for hexameric gap junction connexons derived from gap junctions in vivo. Further, cell-free assembled homo-oligomeric connexons composed of beta1 or beta2 connexin were reconstituted into synthetic lipid bilayers. Single channel conductances were recorded from these bilayers that were similar to those measured for these connexons produced in vivo. Thus, this is the first direct evidence that the synthesis and assembly of a gap junction connexon can take place in microsomal membranes. Finally, the cell-free system has been used to investigate the properties of alpha1, beta1 and beta2 connexin to assemble into hetero-oligomers. Evidence has been obtained for a selective interaction between individual connexin isotypes and that a signal determining the potential hetero-oligomeric combinations of connexin isotypes may be located in the N-terminal sequence of the connexins.

Asunto(s)

Conexinas/biosíntesis , Uniones Comunicantes/metabolismo , Canales Iónicos/biosíntesis , Sistema Libre de Células , Membrana Dobles de Lípidos , Fusión de Membrana , Microsomas/metabolismo , Mutación , Técnicas de Placa-Clamp , Unión Proteica , Biosíntesis de Proteínas , Procesamiento Proteico-Postraduccional , Eliminación de Secuencia

RESUMEN

Electron cryo-microscopy and image analysis of frozen-hydrated, two-dimensional crystals of gap junction membrane channels formed by recombinant alpha 1 connexin (Cx43) reveal a ring of transmembrane alpha-helices that lines the aqueous pore and a second ring of alpha-helices in close contact with the membrane lipids.

Asunto(s)

Conexina 43/química , Animales , Línea Celular , Conexina 43/metabolismo , Cricetinae , Cristalografía por Rayos X , Lípidos de la Membrana/metabolismo , Microscopía Electrónica/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo

RESUMEN

Fatty acid amide hydrolase (FAAH) is a membrane-bound enzyme activity that degrades neuromodulatory fatty acid amides, including oleamide and anandamide. A single 2.5-kb FAAH mRNA is distributed throughout the rat CNS and accumulates progressively between embryonic day 14 and postnatal day 10, remains high until postnatal day 30, then decreases into adulthood. FAAH enzymatic activity, as measured in dissected brain regions, was well correlated with the distribution of its messenger RNA. In situ hybridization revealed profound distribution of FAAH mRNA in neuronal cells throughout the CNS. The most prominent signals were detected in the neocortex, hippocampal formation, amygdala, and cerebellum. The FAAH distribution in the CNS suggests that degradation of neuromodulatory fatty acid amides at their sites of action influences their effects on sleep, euphoria, and analgesia.

Asunto(s)

Amidohidrolasas/análisis , Ácidos Araquidónicos/metabolismo , Sistema Nervioso Central/química , Neuronas/metabolismo , Ácidos Oléicos/metabolismo , ARN Mensajero/análisis , Amidohidrolasas/genética , Animales , Northern Blotting , Endocannabinoides , Hibridación in Situ , Masculino , Alcamidas Poliinsaturadas , Ratas , Ratas Sprague-Dawley

RESUMEN

Oleamide is a sleep-inducing lipid originally isolated from the cerebrospinal fluid of sleep-deprived cats. Oleamide was found to potently and selectively inactivate gap junction-mediated communication between rat glial cells. In contrast, oleamide had no effect on mechanically stimulated calcium wave transmission in this same cell type. Other chemical compounds traditionally used as inhibitors of gap junctional communication, like heptanol and 18beta-glycyrrhetinic acid, blocked not only gap junctional communication but also intercellular calcium signaling. Given the central role for intercellular small molecule and electrical signaling in central nervous system function, oleamide- induced inactivation of glial cell gap junction channels may serve to regulate communication between brain cells, and in doing so, may influence higher order neuronal events like sleep induction.

Asunto(s)

Calcio/metabolismo , Comunicación Celular/efectos de los fármacos , Uniones Comunicantes/fisiología , Neuroglía/metabolismo , Ácidos Oléicos/farmacología , Animales , Línea Celular , Conexina 43/metabolismo , Conexinas/genética , Conexinas/metabolismo , Cricetinae , Colorantes Fluorescentes/metabolismo , Uniones Comunicantes/efectos de los fármacos , Isoquinolinas/metabolismo , Neuroglía/citología , Neuroglía/efectos de los fármacos , Fosforilación , Ratas , Sueño , Relación Estructura-Actividad , Proteína beta1 de Unión Comunicante

RESUMEN

Endogenous neuromodulatory molecules are commonly coupled to specific metabolic enzymes to ensure rapid signal inactivation. Thus, acetylcholine is hydrolysed by acetylcholine esterase and tryptamine neurotransmitters like serotonin are degraded by monoamine oxidases. Previously, we reported the structure and sleep-inducing properties of cis-9-octadecenamide, a lipid isolated from the cerebrospinal fluid of sleep-deprived cats. cis-9-Octadecenamide, or oleamide, has since been shown to affect serotonergic systems and block gap-junction communication in glial cells (our unpublished results). We also identified a membrane-bound enzyme activity that hydrolyses oleamide to its inactive acid, oleic acid. We now report the mechanism-based isolation, cloning and expression of this enzyme activity, originally named oleamide hydrolase, from rat liver plasma membranes. We also show that oleamide hydrolase converts anandamide, a fatty-acid amide identified as the endogenous ligand for the cannabinoid receptor, to arachidonic acid, indicating that oleamide hydrolase may serve as the general inactivating enzyme for a growing family of bioactive signalling molecules, the fatty-acid amides. Therefore we will hereafter refer to oleamide hydrolase as fatty-acid amide hydrolase, in recognition of the plurality of fatty-acid amides that the enzyme can accept as substrates.

Asunto(s)

Amidas/metabolismo , Amidohidrolasas/metabolismo , Ácidos Grasos/metabolismo , Hígado/enzimología , Amidohidrolasas/genética , Amidohidrolasas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Ácidos Araquidónicos/metabolismo , Northern Blotting , Southern Blotting , Células COS , Membrana Celular/enzimología , Cromatografía de Afinidad , Clonación Molecular , Endocannabinoides , Datos de Secuencia Molecular , Alcamidas Poliinsaturadas , Ratas , Homología de Secuencia de Aminoácido